The overall goal of this project continues to be the elucidation of gene/protein systems for novel components of the cutaneous basement membrane zone and the epidermis. This goal is based on the premise that previously uncharacterized proteins are apparently present in the skin, and such proteins could potentially serve as candidate gene/protein systems in different heritable disorders affecting the skin. Thus, information on these new proteins and the corresponding genes is fundamental to comprehensive study of mutations in these diseases. During the past four years of support, we have made major progress in this project. Specifically, we have characterized a number of basement membrane zone genes, and the cDNA and gene probes developed in this project have been instrumental in molecular characterization of mutations in various forms of EB as well as in other disorders affecting the epidermis. An example of such gene/protein systems is the plectin/HD-1, for which we have determined the entire primary sequence by cDNA cloning, we have determined the intron-exon organization of the corresponding gene, and we have performed its chromosomal assignment (1). Furthermore, based on this sequence information we have developed mutation detection strategies, both by heteroduplex scanning and protein truncation tests, which have been successful in identifying a large number of mutations in a specific variant of EB associated with late-onset muscular dystrophy (EB-MD) (2-6). Furthermore, we have identified and characterized several novel genes expressed in the epidermis, including ladinin, a novel BMZ component (7), periplakin, an epidermal envelope protein(8,9), and desmo-15, a desmosomal autoantigen in pemphigus herpetiformis recognized by circulating IgG antibodies in the patients? sera (10). Some of these genes were initially isolated by immunoscreening of cDNA libraries with antibodies from patients with acquired autoimmune blistering diseases. Furthermore, we have identified novel protein-protein interactions by the yeast two-hybrid genetic screen employing a number of BMZ protein domains as baits (see Progress Report). Finally, we have cloned a number of selected mouse cDNA and genomic sequences which have been helpful in development of animal models for EB. An example is cloning of the mouse type VII collagen genomic sequences which were used to construct a "knock-out" vector resulting in the development of a mouse line mimicking human recessive dystrophic EB with extensive blistering phenotype (11, 12). In continuation of this project, we will concentrate our efforts towards completing current studies on three novel epidermal gene/protein systems, viz. ladinin, periplakin, and desmo-15. Secondly, we plan to identify additional novel, previously uncharacterized gene sequences by utilizing immunoscreening methodologies with autoantibodies in patients with acquired forms of blistering skin diseases. Furthermore, we plan to explore the BMZ supramolecular organization by identification and characterization of novel genes which encode interactive proteins, as detected by the yeast-two hybrid genetic screen. Finally, we plan to clone selected mouse cDNA and genomic sequences so as to allow development of animal models for human epidermal diseases.